Assembling, Evolving and Optimizing Hybrid Synthetic Molecular Systems
Synthetic systems provide substantial degrees of freedom, but so far, lack complex functionality. Research methods to efficiently assemble increasingly multifunctional in vitro systems and interface them with hybrid environments to engineer novel, new-to-nature functionalities will be the main goal of this project.
Living cells are usually too complex and their internal network members too interdependent to be tolerant of major functional rearrangements. This project will use the flexibility of compartmentalized synthetic systems to functionally characterize transcriptional and translational signals and provide tools to fine-tune the composition of the cell free extract on which the assembly of the synthetic system is based.
The use of hybrid chemical/biological systems is still a seriously underexplored area. Groundbreaking examples of the flexibility of this approach have been delivered by the group lead by Wolfgang Meier. Other examples include cell free protein synthesis (CFPS), multi-step catalysis, regulation, and minimal cells. Further advances require methods for the controlled assembly of more complex systems and for exploiting the additional flexibility of molecular systems towards new-to-nature (e.g. chemical) system components.
The targeted, complex, multi-component molecular systems are based largely on biological components that need to be recruited and assembled. To prevent laborious one-by-one purification, an inverse strategy will be followed starting with a cell free extract (CFX) from a fully engineered bacterial cell.
In nature, operons are a common bacterial organization principle for functional gene clusters. There, the genes are encoded on a single transcript and protein expression levels are modulated via different control elements. In order to be able to engineer those bacterial systems in a predictive manner, design rules for synthetic multi-gene expression systems need to be established. To achieve the deduction of those rules, we intend to construct thousands of such operons varying different control elements and subsequently quantify protein products and mRNA content. Ultimately, this should allow pre-programming molecular systems at the computer.
As an expansion to on-going efforts to use insulated synthetic molecular systems for preparative chemistry, an approach to optimizing CFPS (Fig. d) will be applied. CFPS is essential in a large variety of evolution efforts that are based on or are supposed to lead to hybrid systems. The focus will be on removing interfering activities such as ATPases, termination factors, and amino acid degrading enzymes while implementing efficient energy regeneration systems.
Later, flexibility of synthetic molecular systems to expand classical protein biochemistry by including non-canonical amino acids into synthesized proteins will be exploited (Fig. e), opening up hosts of possibilities of interfacing novel proteins with synthetic environments (recruitment into membranes, immobilization on functionalized surfaces, complex building) or with novel functionalities (novel cofactors, additional catalytic flexibility).
Read more about the Panke-Group here.